OLIGONUCLEOTIDE CLAMPS HAVING DIAGNOSTIC AND THERAPEUTIC APPLICATIONS

Abstract: Compounds referred to herein as oligonucleotide clamps are provided that stably bind to target polynucleotides in a sequence-specific manner. The oligonucleotide clamps comprise one or more oligonucleotide moieties capable of specifically binding to a target polynucleotide and one or more pairs of binding moieties covalently linked to the oligonucleotide moieties. In accordance with the invention, upon annealing of the oligonucleotide moieties to the target polynucleotide, the binding moieties of a pair are brought into juxtaposition so that they form a stable covalent or non-covalent linkage or complex. The interaction of the binding moieties of the one or more pairs effectively clamps the specifically annealed oligonucleotide moieties to the target polynucleotide.

Description: OLIGONUCLEOTIDE CLAMPS HAVING DIAGNOSTIC AND THERAPEUTIC APPLICATIONS The invention relates generally to oligonucleotides and their use as probes and therapeutic agents, and more particularly, to modified oligonucleotides whose ends are capable of spontaneously forming a stable ring structure whenever such oligonucleotide specifically binds to a target polynucleotide. Background The unpredictability and expense of conventional drug discovery has led to the exploration of several drug discovery approaches that promise more systematic and/or rapid identification candidate compounds for testing in biological assays and disease models. Examples of such approaches include selection of small peptides from a synthetic or recombinant peptide libraries, e.g. Pirrung et al, U.S. patent 5,143,854; Geysen et al, J. Immunol. Meth., 102: 259-274 (1987); Lam et al, Nature, 354: 82-84 (1991); Scott et al, Science, 249: 386-390 (1990); the construction and selection of human or humanized antibodies from recombinant antibody libraries, e.g. Riechmann et al, Nature, 332: 323-327 (1988); Winter and Milstein, Nature, 349: 293-299 (1991); selection of aptamers or ribozymes from random sequence polynucleotide libraries, e.g. Ellington and Szostak, Nature, 346: 818-822 (1990); Blackwell et al, Science, 250: 1104-1110 (1990); Tuerk et al, Science, 249: 505-510 (1990); Joyce, Gene, 82: 83-87 (1989); Cech et al, U.S. patent 4,987,071; Haseloff et al, Nature, 334: 585-591 (1988); and the use of antisense oligonucleotides, e.g. Uhlmann and Peyman, Chemical Reviews, 90: 543-584 (1990); Goodchild, Bioconjugate Chemistry, 1: 165-187 (1990); Helene et al, Biochim. Biophys. Acta, 1049: 99-125 (1990); Cohen, Ed., Oligonucleotides: Antisense Inhibitors of Gene Expression (Macmillan Press, New York, 1989); Crooke, Ann. Rev. Pharmacol. Toxicol., 32: 329-376 (1992); McManaway et al, Lancet, Vol. 335, pgs. 808-811 (1990); Bayever et al, Antisense Research and Development, 2: 109-110 (1992); Manson et al, Lymphokine Research, Vol. 9, pgs. 35-42 (1990); Lisziewicz et al, Proc. Natl. Acad. Sci., 90: 3860-3864 (1993); Miller, Biotechnology, Vol. 9, pgs. 358-362 (1991); Chiang et al, J. Biol. Chem., Vol. 266, pgs. 18162-18171 (1991); Calabretta, Cancer Research, Vol. 51, pgs. 4505-4510 (1991); and the like. Of the cited examples, the antisense approach presents a compelling advantage of not requiring one or more initial screening steps to identify candidate compounds capable of binding to a predetermined target. Specific binding is achieved by providing an oligonucleotide or an analog thereof capable of forming a stable duplex or triplex with a target nucleotide sequence based on Watson-Crick or Hoogsteen binding, respectively. Thus, as soon as the sequence of a target polynucleotide is determined, the structure of candidate antisense compounds is also determined. The specifically bound antisense compound then either renders the respective targets more susceptible to enzymatic degradation, blocks translation or processing, or otherwise blocks or inhibits the function of a target polynucleotide. Another advantage of the antisense approach has been the development of reliable and convenient methods for solid phase synthesis of polynucleotides and analogs thereof, e.g. Caruthers, Science, Vol. 230, pgs 281-285 (1985); Beaucage et al, Tetrahedron, 48: 2223-2311 (1992); and Eckstein, ed., Oligonucleotides and Analogues: A Practical Approach (IRL Press, Oxford, 1991). In particular, the availability of synthetic oligonucleotides and a variety of nuclease-resistant analogs, e.g. phosphorothioates, methylphosphonates, and the like, has encouraged investigation of antisense compounds for treating a variety of conditions associated with the inappropriate expression of indigenous and/or exogenous genes, such as described in the references cited above. Notwithstanding the many hurdles that have been overcome in the course of developing antisense compounds, several significant uncertainties still stand in the way of their widespread adoption as drugs. One such uncertainty concerns the degree of specificity of antisense oligonucleotides under physiological conditions. Antisense oligonucleotides could be non-specific in at least two senses: (i) duplex or triplex formation may lack specificity, e.g. non-perfectly matched duplexes may form— leading to the unwanted inhibition of non-target polynucleotides, and (ii) the moieties not directly involved in base pairing, e.g. the backbone or other appendant groups, may interact non-specifically with other cellular components leading to undesired side effects, e.g. Woolf et al, Proc. Natl. Acad. Sci., 89: 7305-7309 (1992); Matsukura et al, Proc. Natl. Acad. Sci., 84: 7706-7710 (1987); and the like. In regard to first type of nonspecificity, it has been observed that duplexes involving longer oligonucleotides tend to be more tolerant of mismatches-and hence, less specific-than duplexes involving shorter oligonucleotides, e.g. Young et al, Nucleic Acids Research, 19: 2463-2470 (1991). In regard to the second type of nonspecificity, such activity may not be surprising in view of the large body of work on the use of polyanions, in particular homopolymeric polynucleotides, as anti-viral compounds, e.g. Levy, Chapter 7, in Stringfellow, editor, Interferon and Interferon Inducers (Marcel Dekker, New York, 1980). Interestingly, increased activity—and with some polyanions increased toxicity— was observed with increased polymer size. The uncertainty over nonspecific binding has led to the exploration of several ways to modify oligonucleotides to enhance duplex or triplex stability of antisense compounds. One approach has been to couple duplex or triplex intercalating moieties to the antisense oligonucleotide, e.g. Park et al, Proc. Natl. Acad. Sci., 89: 6653-6657 (1992); Stein et al, Gene, 72: 333-341 (1988); Mergny et al, Science, 256: 1681-1684 (1992); Miller, International application PCT/US92/ 03999; and the like. Another approach involves the use of circular oligonucleotides, which are exonuclease resistant and have been shown to melt from single-stranded targets at substantially higher temperatures than linear oligonucleotides when binding involves both Watson-Crick and Hoogsteen base pairing, e.g. Prakash and Kool, J. Am. Chem. Soc, 114: 3523-3527 (1992). Additional approaches for enhancing specificity and binding strength would be highly useful for DNA-based therapeutics and diagnostic applications of nucleic acids. Summary of the Invention The invention relates to compounds capable of forming stable circular complexes and/or covalently closed macrocycles after specifically binding to a target polynucleotide. Generally, compounds of the invention comprise one or more oligonucleotide moieties capable of specifically binding to a target polynucleotide and one or more pairs of binding moieties covalently linked to the oligonucleotide moieties. In accordance with the invention, upon annealing of the oligonucleotide moieties to the target polynucleotide, the binding moieties of a pair are brought into juxtaposition so that they form a stable covalent or non-covalent linkage or complex. The interaction of the binding moieties of die one or more pairs effectively clamps the specifically annealed oligonucleotide moieties to die target polynucleotide. In one aspect, compounds of the invention comprise a first binding moiety, a first oligonucleotide moiety, a hinge region, a second oligonucleotide moiety, and a second binding moiety, for example, as represented by the particular embodiment of the following formula: X-O,-G-O 2 -Y wherein θ ! and O 2 are the first and second oligonucleotide moieties, G is die hinge region, X is the first binding moiety and Y is die second binding moiety such that X and Y form a stable covalent or non-covalent linkage or complex whenever they are brought into juxtapositon by the annealing of die oligonucleotide moieties to a target polynucleotide, as illustrated diagrammatically in Figure la. Preferably, in this embodiment, one of Oj and O 2 forms a duplex mrough Watson-Crick type of binding widi the target polynucleotide while the other of O. and O 2 forms a triplex through Hoogsteen or reverse Hoogsteen type of binding. Whenever X and Y form a covalent linkage, the compound of the invention forms a macrocycle of the following form: wherein "XY" is the covalent linkage formed by the reaction of X and Y. In another aspect, compounds of the invention comprise a first binding moiety, a first, second, and diird oligonucleotide moiety, a first and second hinge region, and a second binding moiety, for example, as represented by the particular embodiment of the following formula: X-O.-G r O 2 -G 2 -O 3 -Y wherein X and Y are described as above, G. and G 2 are the first and second hinge regions, and O., O 2 , and O 3 are the first through third oligonucleotide moieties. Preferably, die sequences of O l5 O 2 , and O 3 are selected so that 0_ and O 2 and O 3 and O 2 form triplex structures with the target polynucleotide, as diagrammatically illustrated in Figure lb. Whenever X and Y form a covalent linkage, the compound of d e invention forms a macrocycle of the following form: wherein "XY" is the covalent linkage formed by the reaction of X and Y. In yet another aspect, the oligonucleotide clamps of die invention are compositions of two or more components, e.g. having the form: X-O.-W and Y-O 2 -Z wherein X, Y, W, and Z are defined as X and Y above. In tiiis embodiment, me hinge region is replaced by additional complex-forming moieties W and Z. As above, one of Oj and O 2 undergoes Watson-Crick type of binding while die otiier undergoes Hoogsteen or reverse Hoogsteen type of binding to a target polynucleotide, as shown diagrammatically in Figure lc. Similarly, whenever X and Y and W and Z form covalent linkages, compounds X-O.-W and Y-O 2 -Z form a macrocycle of the following form: OR depending on die selection of O* and O 2 . Preferably, compounds of die invention are capable of forming covalently closed macrocycles or stable circular complexes topologically linked to a target polynucleotide. The invention provides compounds capable of specifically binding to predetermined target polynucleotides widi superior stability than currently available probes and antisense compounds. Compounds of die invention are employed eidier as antisense or anti-gene compounds to inhibit the function of a polynucleotide whose expression or participation in a regulatory function is associated widi a disease state or as probes for detecting die presence of a target polynucleotide. The invention includes die oligonucleotide clamps per se as well as pharmaceutical compositions and kits for particular applications. Brief Description of die Figures Figures la-c diagrammatically illustrate how three separate embodiments of die invention bind or "clamp" a target polynucleotide. Figure 2a-b diagrammatically illustrates the concept of "topologically linked." Figure 3 illustrates the results of the inhibition of die cytotoxic effects of HIV in an ATH8 cell assay. Definitions "Topologically linked" in reference to compounds of e invention refers to the relative configuration of target polynucleotide and oligonucleotide clamp wherein the oligonucleotide clamp forms a closed circular complex or macrocycle enclosing the target polynucleotide strand, as shown diagrammatically in Figures 2a and 2b. In Figure 2a, oligonucleotide clamp 20 is topologically linked to target polynucleotide 10. In Figure 2b, oligonucleotide 20 is topologically disjoint from target polynucleotide 10. "Stable" in reference to die formation of a covalent linkage and/or non-covalent complex between binding moieties means mat melting temperature of the oligonucleotide clamp incorporating the given pair(s) of binding moieties and its target polynucleotide is increased by at least fifty percent over the melting temperature of oligonucleotide moieties of die clamp alone, wherein melting temperature is measured by standard techniques, e.g. half maximum of 260 nm absorbance v. temperature as described more fully below. "Linkage" in reference to the reaction of binding moieties includes bodi covalent linkages and non-covalent complexes. The term "oligonucleotide" as used herein includes linear oligomers of natural or modified monomers or linkages, including deoxyribonucleosides, ribonucleosides, -anomeric forms thereof, polyamide nucleic acids, and the like, capable of specifically binding to a target polynucleotide by way of a regular pattern of monomer-to-monomer interactions, such as Watson-Crick type of base pairing, Hoogsteen or reverse Hoogsteen types of base pairing, or the like. Usually monomers are linked by phosphodiester bonds or analogs diereof to form oligonucleotides ranging in size from a few monomeric units, e.g. 3-4, to several hundreds of monomeric units. Whenever an oligonucleotide is represented by a sequence of letters, such as "ATGCCTG," it will be understood diat the nucleotides are in 5'-> 3' order from left to right and that "A" denotes deoxyadenosine, "C" denotes deoxycytidine, "G" denotes deoxyguanosine, and "T" denotes thymidine, unless otherwise noted. Analogs of phosphodiester linkages include phosphorodiioate, phosphorodlthioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilo iioate, phosphoranilidate, phosphoramidate, and d e like. As used herein, "nucleoside" includes the natural nucleosides, including 2'-deoxy and 2'-hydroxyl forms, e.g. as described in Kornberg and Baker, DNA Replication, 2nd Ed. (Freeman, San Francisco, 1992). "Analogs" in reference to nucleosides includes syndietic nucleosides having modified base moieties and/or modified sugar moieties, e.g. described generally by Scheit, Nucleotide Analogs (John Wiley, New York, 1980). Such analogs include synthetic nucleosides designed to enhance binding properties, e.g. stability, specificity, or the like, such as disclosed by . Detailed Description of die Invention The invention relates to oligonucleotide clamps mat are capable of binding to a specific region of a target polynucleotide. The clamping aspect of the compounds is achieved by die formation of stable linkages or complexes between binding moieties after they are brought into proximity by specific binding of the one or more oligonucleotide moieties to a target polynucleotide. Preferably, oligonucleotide moieties of die compounds of die invention are selected so diat iey simultaneously undergo Watson-Crick and Hoogsteen types of binding widi specific regions of a target polynucleotide. In embodiments where triplex formation is desired, diere are constraints on the selection of target sequences. Generally, third strand association via Hoogsteen type of binding is most stable along homopyrimidine -homopurine tracks in a double stranded target. Usually, base triplets form in T-A*T or C-G*C motifs (where "-" indicates Watson-Crick pairing and "*" indicates Hoogsteen type of binding); however, odier motifs are also possible. For example, Hoogsteen base pairing permits parallel and antiparallel orientations between the third strand (die Hoogsteen strand) and die purine-rich strand of die duplex to which die third strand binds, depending on conditions and die composition of die strands. There is extensive guidance in die literature for selecting appropriate sequences, orientation, conditions, nucleoside type (e.g. whed er ribose or deoxyribose nucleosides are employed), base modifications (e.g. methylated cytosine, and d e like) in order to maximize, or odierwise regulate, triplex stability as desired in particular embodiments, e.g. Roberts et al, Proc. Natl. Acad. Sci., 88: 9397-9401 (1991); Roberts et al, Science, 258: 1463-1466 (1992); Distefano et al, Proc. Natl. Acad. Sci., 90: 1179-1183 (1993); Mergny et al, Biochemistry, 30: 9791-9798 (1991); Cheng et al, J. Am. Chem. Soc, 114: 4465-4474 (1992); Beal and Dervan, Nucleic Acids Research, 20: 2773-2776 (1992); Beal and Dervan, J. Am. Chem. Soc, 114: 4976-4982 (1992); Giovannangeli et al, Proc. Nad. Acad. Sci., 89: 8631-8635 (1992); Moser and Dervan, Science, 238: 645-650 (1987); McShan et al, J. Biol. Chem., 267:5712-5721 (1992); Yoon et al, Proc. Natl. Acad. Sci., 89: 3840-3844 (1992); Blume et al, Nucleic Acids Research, 20: 1777-1784 (1992); and the like. Generally, after one of the oligonucleotide moieties forms a Watson-Crick duplex widi a pyrimidine-rich or purine-rich track in a target polynucleotide, the remaining oligonucleotide components bind to die major groove of the duplex to form a triplex structure. Selection of particular oligonucleotide sequences for triplex formation can also be carried out empirically, for example, through aptamer screening, or like process, where candidate oligonucleotide moieties are selected on die basis of binding strength to an immobilized double stranded target, e.g. Ellington and Szostak, Nature, 346: 818-822 (1990); Toole et al, International application PCT/US92/01383; and the like. Target polynucleotides may be single stranded or double stranded DNA or RNA; however, single stranded DNA or RNA target polynucleotides are preferred. Preferably, stability of oligonucleotide clamp/target polynucleotide complexes are determined by way of melting, or strand dissociation, curves. The temperature of fifty percent strand dissociation is taken as die melting temperature, T m , which, in turn, provides a convenient measure of stability. T m measurements are typically carried out in a saline solution at neutral pH widi target and clamp concentrations at between about 1.0-2.0 μM. Typical conditions are as follows: 150 mM NaCl and 10 mM MgCl 2 in a 10 mM sodium phosphate buffer (pH 7.0) or in a 10 mM Tris-HCl buffer (pH 7.0); or like conditions. Data for melting curves are accumulated by heating a sample of die oligonucleotide clamp/target polynucleotide complex from room temperature to about 85-90°C. As the temperature of the sample increases, absorbance of 260 nm light is monitored at 1°C intervals, e.g. using a Cary (Australia) model IE or a Hewlett-Packard (Palo Alto, CA) model HP 8459 UV/VIS spectrophotometer and model HP 89100A temperature controller, or like instruments. The oligonucleotide moieties of die invention are syndiesized by conventional means on a commercially available automated DNA syndiesizer, e.g. an Applied Biosystems (Foster City, CA) model 380B, 392 or 394 DNA/RNA syndiesizer. Preferably, phosphoramidite chemistry is employed, e.g. as disclosed in die following references: Beaucage and Iyer, Tetrahedron, 48: 2223-2311 (1992); Molko et al, U.S. patent 4,980,460; Koster et al, U.S. patent 4,725,677; Caruthers et al, U.S. patents 4,415,732; 4,458,066; and 4,973,679; and die like. For dierapeutic use, nuclease resistant backbones are preferred. Many types of modified oligonucleotides are available iat confer nuclease resistance, e.g. phosphorodiioate, phosphorodidiioate, phosphoramidate, or die like, described in many references, e.g. phosphorothioates: Stec et al, U.S. patent 5,151,510; Hirschbein, U.S. patent 5,166,387; Bergot, U.S. patent 5,183,885; phosphoramidates: Froehler et al, International application PCT/US90/03138; and for a review of additional applicable chemistries: Uhlmann and Peyman (cited above). In some embodiments it may be desirable to employ P-chiral linkages, in which case die chemistry disclosed by Stec et al, European patent application 92301950.9, may be appropriate. The length of the oligonucleotide moieties is sufficiently large to ensure tiiat specific binding will take place only at the desired target polynucleotide and not at odier fortuitous sites. The upper range of the lengdi is determined by several factors, including the inconvenience and expense of synthesizing and purifying oligomers greater than about 30-40 nucleotides in length, die greater tolerance of longer oligonucleotides for mismatches than shorter oligonucleotides, and die like. Preferably, the oligonucleotide moieties have lengdis in the range of about 6 to 40 nucleotides. More preferably, die oligonucleotide moieties have lengdis in die range of about 12 to 25 nucleotides. Hinge regions consist of nucleosidic or non -nucleosidic polymers which preferably facilitate die specific binding of die monomers of die oligonucleotide moieties widi tiieir complementary nucleotides of die target polynucleotide. Generally, die oligonucleotide moieties may be connected to hinge regions and/or binding moieties in eidier 5'-> 3' or 3'->5' orientations. For example, in die embodiment described above comprising a first binding moiety, a first oligonucleotide moiety, a hinge region, a second oligonucleotide moiety, and a second binding moiety, die oligonucleotide moieties may have any of die following orientations: X-(5')N I N 2 N 3 - ... -N j (3')-G-(5')N 1 N 2 N 3 - ... -N k (3')-Y OR X-(5')N,N 2 N 3 - ... -N j (3')-G-(3')N k N k . 1 N k . 2 - ... -N.(5')-Y OR ... -N 1 (5')-G-(5')N 1 N 2 N 3 - ... -N k (3')-Y OR X-O N j N^N j. ,- ... -N 1 (5')-G-(3')N k N k.1 N k.2 - ... - Nl (5')-Y wherein N,N 2 N 3 - ... -N and NjN 2 N 3 - ... -N j are k-mer and j-mer oligonucleotide moieties in the indicated orientations. Preferably, the hinge region has the general form: (M-L) n wherein M may be an inert non -sterically hindering spacer moiety serving to connect the oligonucleotide moieties, wherein die M 's and L 's in any given chain may be e same or different. Alternatively, one or more of monomers M may contain reactive functionalities for attaching labels; oligonucleotides or odier binding polymers for hybridizing or binding to amplifier strands or structures, e.g. as described by Urdea et al, U.S. patent 5,124,246 or Wang et al, U.S. patent 4,925,785; "hooks", e.g. as described in Whiteley et al, U.S. patent 4,883,750; or odier groups for affecting solubility, cellular delivery, promotion of duplex and/or triplex formation, such as intercalators, alkylating agents, and die like. Preferably, the hinge regions provide a spacer of about 16-28 angstroms between the termini of the oligonucleotides. Preferably, L is a phosphorus(V) linking group which may be phosphodiester, phosphotriester, methyl or ethyl phosphonate, phosphorodiioate, phosphorodidiioate, phosphoramidate, or die like. Generally, linkages derived from phosphoramidite precursors are preferred so mat compounds of die invention can be conveniently syndiesized widi commercial automated DNA syndiesizers, e.g. Applied Biosystems, Inc. (Foster City, CA) model 394, or the like. n may vary significantly depending on the nature of M and L. Generally, n will vary from 1 for M comprising alkyl, alkenyl, and/or ediers containing 10 or more carbon atoms, e.g. Salunkhe et al, J. Am. Chem. Soc, 114: 8768-8772 (1992), to about 10 for M comprising alkyl, alkenyl, and/or ediers containing 2-3 carbon atoms. Preferably, for a hinge moiety consisting entirely of an alkyl chain (and linkage moieties), such alkyl chain contains form 8 to 15 carbon atoms, and more preferably, from 9 to 12 carbon atoms. Preferably, for nucleoside-sized monomers, n varies between about 3 and about 10; and more preferably, n varies between about 4 and 8. Preferably, hinge moieties are syndiesized using conventional phosphoramidite and/or hydrogen phosphonate chemistries. The following references disclose several phosphoramidite and/or hydrogen phosphonate monomers suitable for use in die present invention and provide guidance for their syndiesis and inclusion into oligonucleotides: Newton et al, Nucleic Acids Research, 21: 1155-1162 (1993); Griffin et al, J. Am. Chem. Soc, 114: 7976-7982 (1992); Jaschke et al, Tetrahedron Letters, 34: 301-304 (1992); Ma et al, International application PCT/CA92/00423; Zon et al, International application PCT/US90/06630; Durand et al, Nucleic Acids Research, 18: 6353-6359 (1990); Salunkhe et al, J. Am. Chem. Soc, 114: 8768-8772 (1992); and the like. In a preferred embodiment, M is a straight chain, cyclic, or branched organic molecular structure containing from 1 to 20 carbon atoms and from 0 to 10 heteroatoms selected from the group consisting of oxygen, nitrogen, and sulfur. More preferably, M is alkyl, alkoxy, alkenyl, or aryl containing from 1 to 16 carbon atoms; heterocyclic having from 3 to 8 carbon atoms and from 1 to 3 heteroatoms selected from the group consisting of oxygen, nitrogen, and sulfur; glycosyl; or nucleosidyl. Most preferably, M is alkyl, alkoxy, alkenyl, or aryl containing from 1 to 8 carbon atoms; glycosyl; or nucleosidyl. A variety of binding moieties are suitable for use with die invention. Generally, they are employed in pairs, which for convenience here will be referred to as X and Y. X and Y may be die same or different. Whenever die interaction of X and Y is based on die formation of stable hydrophobic complex, X and Y are lipophilic groups, including alkyl groups, fatty acids, fatty alcohols, steroids, waxes, fat-soluble vitamins, and die like. Further exemplary lipophilic binding moieties include glycerides, glyceryl ediers, phospholipids, sphingolipids, terpenes, and the like. In such embodiments, X and Y are preferably selected from die group of steroids consisting of a derivatized perhydrocyclopentanophenandirene nucleus having from 19 to 30 carbon atoms, and 0 to 6 oxygen atoms; alkyl having from 6 to 16 carbon atoms; vitamin E; and glyceride having 20 to 40 carbon atoms. Preferably, a perhydrocyclopentanophenanthrene-based moiety is attached dirough the hydroxyl group, eidier as an ether or an ester, at its C3 position. It is understood dia X and Y may include a linkage group connecting it to an oligonucleotide moiety. For example, glyceride includes phosphoglyceride, e.g. as described by MacKellar et al, Nucleic Acids Research, 20: 3411-3417 (1992), and so on. It is especially preferred diat lipophilic moieties, such as perhydrocyclopentanophenandirene derivatives, be linked to the 5' carbon and/or the 3' carbon of an oligonucleotide moiety by a short but flexible linker that permits die lipophilic moiety to interact widi die bases of die oligonucleotide clamp/target polynucleotide complex or a lipophilic moiety on the same or another oligonucleotide moiety. Such linkers include phosphate (i.e. phosphodiester), phosphoramidate, hydroxyurediane, carboxyaminoalkyl and carboxyaminoalkylphosphate linkers, or the like. Preferably, such linkers have no more an from 2 to 8 carbon atoms. Binding moieties can be attached to die oligonucleotide moiety by a number of available chemistries. Generally, it is preferred d at die oligonucleotide be initially derivatized at its 3' and/or 5' terminus widi a reactive functionality, such as an amino, phosphate, thiophosphate, or thiol group. After derivatization, a hydrophilic or hydrophobic moiety is coupled to die oligonucleotide via die reactive functionality. Exemplary means for attaching 3' or 5' reactive functionalities to oligonucleotides are disclosed in Fung et al, U.S. patent 5,212,304; Connolly, Nucleic Acids Research, 13: 4485-4502 (1985); Tino, International application PCT/US91/09657; Nelson et al, Nucleic Acids Research, 17: 7187-7194 (1989); Stabinsky, U.S. patent 4,739,044; Gupta et al, Nucleic Acids Research, 19: 3019 (1991); Reed et al, International application PCT/US91/06143; Zuckerman et al, Nucleic Acids Research, 15: 5305 (1987); Eckstein, editor, Oligonucleotides and Analogues: A Practical Approach (IRL Press, Oxford, 1991); Clontech 1992/1993 Catalog (Clontech Laboratories, Palo Alto, CA); and like references. Preferably, whenever X and Y form a covalent linkage, X and Y pairs must react specifically widi each odier when brought into juxtaposition, but otherwise diey must be substantially unreactive with chemical groups present in a cellular environment. In this aspect of the invention, X and Y pairs are preferably selected from the following group: when one of X or Y is phosphorodiioate, die odier is haloacetyl, haloacyl, haloalkyl, or alkylazide; when one of X or Y is thiol, the odier is alkyl iodide, haloacyl, or haloacetyl; when one of Y or Y is phenylazide die odier is phenylazide. More preferably, when one of X or Y is phosphorodiioate, die other is haloacetyl, haloacyl, or haloalkyl, wherein said alkyl, acetyl, or acyl moiety contains from one to eight carbon atoms. Most preferably, when one of X or Y is phosphorodiioate, the other is haloacetyl. Most preferably, whenever one of X or Y is phosphorodiioate, the other is bromoacetyl. These binding moieties form a covalent diiophosphoylacetylamino bridge, as shown below, selectively and efficiently at low concentrations, e.g. less dian one μM, when reacted in an aqueous environment in the presence of a target polynucleotide: 5'- N,N 2 N 3 ... N r NHC(=O)CH 2 X + S-P(=O)(O-)O-N.N 2 N 3 ... N k -3' — > 1 2 N,N 2 N 3 ... N r NHC(=O)CH 2 SP(=O)(O-)O-N.N 2 N 3 ... N k wherein X is halo and N„ N 2 , ... N- and N k are nucleotides of a j-mer and k-mer, respectively. Compound 1 can be prepared by N-succinimidyl haloacetate in N,N-dimethylformamide (DMF) with a 3 '-aminodeoxyribonucleotide precursor in a sodium borate buffer at room temperature. After about 35 minutes the mixture is diluted (e.g. widi H 2 O), desalted and, purified, e.g. by reverse phase HPLC. The 3 '-aminodeoxyribonucleotide precursor can be prepared as described in Gryaznov and Letsinger, Nucleic Acids Research, 20: 3403-3409 (1992) or Tetrahedron Letters, 34: 1261-1264 (1993). Briefly, after deprotection, the 5' hydroxyl of a deoxydiymidine linked to a support via a standard succinyl linkage is phosphitylated by reaction widi chloro-(diisopropyledιylamino) -medioxyphosphine in an appropriate solvent, such as dichloromediane/diisopropylediylamine. After activation widi tetrazole, die 5'-phosphitylated thymidine is reacted with a 5'-trityl-O-3'-amino-3'-deoxynucleoside to form a nucleoside-diymidine dimer wherein die nucleoside moieties are covalently joined by a phosphoramidate linkage. The remainder of die oligonucleotide is syndiesized by standard phosphoramidite chemistry. After cleaving the succinyl linkage, the oligonucleotide widi a 3' terminal amino group is generated by cleaving the phosphoramidate link by acid treatment, e.g. 80% aqueous acetic acid for 18-20 hours at room temperature. 5'-trityl-O-3'-amino-3'-deoxynucleosides may be syndiesized in accordance with Glinski et al, J. Chem. Soc. Chem. Comm., 915-916 (1970); Miller et al, J. Org. Chem. 29: 1772 (1964); Zielinki and Orgel, Nucleic Acids Research, 13: 2469-2484 (1985) and 15: 1699-1715 (1987); Ozols et al, Synthesis, 7: 557-559 (1980); and Azhayev et al, Nucleic Acids Research, 6: 625-643 (1979); which references are incorporated by reference. 5' monophosphorodiioate 2 is formed as follows: A 5' monophosphate is attached to die 5' end of an oligonucleotide eidier chemically or enzymatically with a kinase, e.g. Sambrook et al, Molecular Cloning: A Laboratory Manual, 2nd Edition (Cold Spring Harbor Laboratory, New York, 1989). Preferably, as a final step in oligonucleotide syndiesis, a monophosphate is added by chemical phosphorylation as described by Horn and Urdea, Tetrahedron Lett., 27: 4705 (1986) (e.g. using commercially available reagents such as 5' Phosphate-ON™ from Clontech Laboratories (Palo Alto, California)). The 5'-monophosphate is then sulfurized using conventional sulfurizing agents, e.g. treatment widi a 5% solution of S 8 in pyridine/CS 2 (1:1, v/v, 45 minutes at room temperature); or treatment with sulfurizing agent described in U.S. patents 5,003,097; 5,151,510; or 5,166,387. Preferably, the haloacetylamino derivatized oligonucleotides are synthesized separately from unprotected monophosphorodiioate groups. Compounds of die invention can be employed as diagnostic probes to detect die presence of one or more target polynucleotides in a wide range of samples, including environmental samples, e.g. from public water supplies, samples from foodstuffs, and from odier biological samples, such as blood, saliva, semen, amniotic fluid, tissue homogenates of plants or animals, or of human patients, and die like. The use of nucleic acid probes in human diagnostics, forensics, and genetic analysis has been extensively reviewed. For example, die following references describe many diagnostic applications of nucleic acid probes for which the present invention can be usefully employed: Caskey, Science 236: 1223-1228 (1987); Landegren et al, Science, 242: 229-237 (1988); and Arnheim et al, Ann. Rev. Biochem., 61: 131-156 (1992). Moreover, there is extensive guidance in die literature concerning die selection of hybridization conditions, labeling means, and die like, which is applicable to die practice of the present invention, e.g. Wallace et al, Nucleic Acids Research 6: 3543-3557 (1979); Crodiers et al, J. Mol. Biol. 9: 1-9 (1964); Gotoh, Adv. Biophys. 16: 1-52 (1983); Wetmer, Critical Reviews in Biochemistry and Molecular Biology 26: 227-259 (1991); Breslauer et al, Proc. Natl. Acad. Sci. 83: 3746-3750 (1986); Wolf et al, Nucleic Acids Research, 15: 2911-2926 (1987); McGraw et al, Biotechniques, 8: 674-678 (1990), and the like. Oligonucleotide clamps of die invention may be used in essentially any of die known solution or solid phase hybridization formats, such as those in which the analyte is bound directly to a solid phase, or sandwich hybridizations in which die analyte is bound to an oligonucleotide diat is, in turn, bound to a solid phase. Oligonucleotide clamps having an oligonucleotide "tail" attached to a hinge region are particularly useful in conjunction widi branched polymer amplification schemes, such as mose disclosed by Urdea et al, U.S. patent 5,124,246; Wang et al, U.S. patent 4,925,785; and the like. Urdea et al and Wang et al are incorporated by reference for their description of such hybridization assays. In such embodiments, d e oligonucleotide clamp serves as a highly stable "capture" probe by binding to a target polynucleotide analyte of interest. The oligonucleotide tail then hybridizes widi a directiy or indirectly labeled amplifier strand or complex. Such tails are long enough to form a stable duplex widi die amplifier strand. Preferably, such tails are between 18 and 60 nucleotides in length. Tails may also comprise a second oligonucleotide clamp. That is, a dimer of oligonucleotide clamps having different binding specificities can be used to tightly couple an amplifier complex to a target polynucleotide. Preferably, oligonucleotide tails are coupled to hinge regions at an amino group which has been derivatized with bromoacetyl. An oligonucleotide tail having either a 5' or 3' phosphorodiioate group is then reacted with the bromoacetyl group to form a thiophosphorylacetylamino bridge, as described more fully above and in the Examples below. Phosphoramidate linkages are introduced in accordance with published procedures, e.g. Letsinger, U.S. patent 4,958,013; Agrawal et al, Nucleic Acids Research, 18: 5419-5423 (1990); or die like. By a similar procedure, dimers of oligonucleotide clamps can also be constructed. Whenever oligonucleotide clamps of the invention are employed in diagnostic assays, or in odier processes not requiring direct contact widi a patient, a wider range of binding moieties may be employed than would be possible for therapeutic use. In diagnostic and odier non -tiierapeutic applications, reaction of die binding moieties may involve an activation step wherein one or botii of the binding moieties are activated or rendered reactive towards one anodier by exposure to an activating agent or condensing agent, such as radiation, a reducing agent, an oxidizing agent, or die like. Exemplary, binding moieties employing activating agents include tiiiophosphoryl groups in die presence of K 3 Fe(CN) 6 or KI 3 , e.g. Gryaznov and Letsinger, Nucleic Acids Research, 21: 1403-1408 (1993); phosphoryl and hydroxyl in die presence of N-cyanoimidazole, e.g. Luebke et al, J. Am. Chem. Soc, 113: 7447-7448 (1991); phosphoryl or amino group and hydroxyl in die presence of cyanogen bromide, e.g. Sokolova et al, FEBS Letters, 232: 153-155 (1988); phosphoryl and hydroxyl groups in die presence of spermine-5-(N-edιylimidazole)carboxamide and cyanoimidazole, e.g. Zuber et al, J. Am. Chem. Soc, 115: 4939-4940 (1993); and the like. Kits incorporating oligonucleotide clamps can take a variety of forms depending on die particular embodiment, die type of assay format employed, and die labeling scheme employed. Generally, kits of the invention comprise an oligonucleotide clamp specific for a given target polynucleotide, a hybridization buffer, and a signal generation moiety. Kits of die invention may further comprise wash buffers for removing unbound label and/or oligonucleotide clamps, solid phase supports such as derivatized magnetic beads, or die like; and prehybridization buffers containing blocking agents, e.g. Denhardt's solution, sonicated salmon sperm DNA, detergents such as 1% SDS, or the like, for minimizing nonspecific binding of oligonucleotide clamps or odier nucleosidic binding components, such as amplifier strands. An exemplary hybridization buffer comprises the following reagents: 100-150 mM NaCl, 10 mM MgCl 2 , and 10 mM Tris-HCl (pH 7.0). Signal generation moieties are molecular structures that directly or indirectly generate a signal, e.g. fluorescent, colorimetric, radioactive, or the like, that can be detected by conventional means. Direct signal generation means at die moiety producing a signal is covalently linked to die oligonucleotide clamp, e.g. as with the covalent attachment of a fluorescent dye, enzyme, or the like. Indirect signal generation means at a particular moiety, e.g. an oligonucleotide tail on a hinge region, is one component of a multi-component system at produces a signal. Preferably, die signal generation moieties comprises an oligonucleotide tail of about 12 to about 50 nucleotides in lengtii covalently attached to a hinge region of an oligonucleotide clamp. In one aspect of this preferred embodiment, a signal is generated indirectly by providing a second oligonucleotide which is complementary to the tail and which has a fluorescent dye covalently attached. Attaching fluorescent dyes to oligonucleotides is well known in die art, e.g. U.S. patents 4,997,828; 5,151,507; 4,855,225; 5,188,934; Eckstein, editor (cited above); and the like. Compounds of die invention can be employed as components of pharmaceutical compositions. A variety of diseases and disorders can be treated by administration of a composition comprising compounds of the invention. Viral diseases that can be treated by antisense inhibition of nucleic acid expression include, but are not limited to, those caused by hepatitis B virus, cytomegalovirus, herpes simplex virus I or II, human immunodeficiency virus type I or II, influenza virus, respiratory syncytial virus, and human papilloma virus. Malignancies which can be treated by administration of antisense compounds of die invention include tiiose known to be directly or indirectly caused by die inappropriate expression of one or more gene, such as cancers caused by die inappropriate expression of oncogenes, e.g. myb, bcr-abl, kit, myc, ras, raf, abl, or die like. In such diseases, e compounds of die invention are specifically targeted to aberrantly expressed genes associated widi die diseases, or to regulatory polynucleotides diat interact with aberrantly transcribed or expressed genes, e.g. Aaronson, Science, Vol. 254, pgs. 1146-1153 (1991). Acute inflammatory and immune reactions, such as septic shock, eosinophilia, and die like, can also be treated with compounds of die invention, wherein inappropriately and/or aberrantly expressed cytokine genes are inhibited, e.g. Tracey et al, Nature, Vol. 330, pgs. 662-664 (1987), U.S. patent 5,055,447, and Waage et al, J. Exp. Med., Vol. 169, pgs. 333-338 (1989)(antisense TNF-or and/or TNF-/5); Starnes et al, J. Immunol., Vol. 145, pgs. 4185-4191 (1990), and Fong et al, J. Immunol., Vol. 142, pgs. 2321-2324 (antisense IL-6); Coffman et al, Science, Vol.245 pgs. 308-310 (antisense IL-5); Finkelman et al, J. Immunol., Vol. 141, pgs. 2335-2341 (1988)(antisense IL-4); Young et al, Blood, Vol. 68, pgs. 1178-1181 (1986)(antisense GM-CSF); and the like. The components included in pharmaceutical compositions of die invention depend on several factors, including die nature of die disease or condition being treated, die location of disease lesions, die mode of drug delivery and/or administration contemplated, die latter of which can include in vivo administration by way of regional or systemic perfusion, topical application, intranasal administration, administration by implanted or transdermal sustained release systems, and die like, as well as ex vivo administration for use in bone marrow purging. A preferred metiiod of administration of oligonucleotide clamps comprises eitiier regional or systemic perfusion. According to a metiiod of regional perfusion, the afferent and efferent vessels supplying the extremity containing a lesion, e.g. a cancerous lesion, are isolated and connected to a low-flow perfusion pump in continuity with an oxygenator and heat exchanger. The iliac vessels may be used for perfusion of the lower extremities. The axillary vessels are cannulated high in the axilla for upper extremity lesions. A pharmaceutical composition containing an oligonucleotide clamp is added to die perfusion circuit, and die perfusion is continued for an appropriate time period, e.g. an hour. Perfusion rates of from 100 to 150 ml/minute may be employed for lower extremity lesions, while half that rate should be employed for upper extremity lesions. Systemic heparinization may be used diroughout the perfusion, and reversed after d e perfusion is complete. This isolation perfusion technique permits administration of higher dosed of chemotiierapeutic agent than would otherwise be tolerated upon infusion into the arterial or venous systemic circulation. For systemic infusion, the oligonucleotide clamps are preferably delivered via a central venous catheter, which is connected to an appropriate continuous infusion device. Indwelling catiieters provide long term access to the intravenous circulation for frequent administrations of drugs over extended periods of time. Generally a pharmaceutical composition of die invention facilitates die delivery of an effective amount of the active drug to a desired site in a manner consistent widi patient safety and comfort. An effective amount of an oligonucleotide clamp depends on several factors, including die disease or condition being treated, die method of administration, die scheduling of die administration, die condition of die patient, and d e like. Typically, a parentially administered dose will be in die range of about lg/kg/day to about 100 mg/kg/day of patient body weight. A key factor in selecting an appropriate dose for a given condition or disease is die dierapeutic result, as measure by standard criteria well known to the medical practitioner, e.g. for oncological applications see: Cancer: Principles and Practice of Oncology, 3rd Edition, edited by V.T. DeVita et al (Lippincott Company, Philadelphia, 1989). Pharmaceutical compositions of the invention include a pharmaceutical carrier that may contain a variety of components that provide a variety of functions, including regulation of drug concentration, regulation of solubility, chemical stabilization, regulation of viscosity, absorption enhancement, regulation of pH, and die like. For example, in water soluble formulations tiie pharmaceutical composition preferably includes a buffer such as a phosphate buffer, or other organic acid salt, preferably at a pH of between about 7 and 8. For formulations containing weakly soluble oligonucleotide clamps, microemulsions may be employed, for example by using a nonionic surfactant such as Tween 80 in an amount of 0.04-0.05% (w/v), to increase solubility. Other components may include antioxidants, such as ascorbic acid, hydrophilic polymers, such as, monosaccharides, disaccharides, and other carbohydrates including cellulose or its derivatives, dextrins, chelating agents, such as EDTA, and like components well known to tiiose in die pharmaceutical sciences, e.g. Remington's Pharmaceutical Science, latest edition (Mack Publishing Company, Easton, PA). Compounds of die invention include die pharmaceutically acceptable salts thereof, including tiiose of alkaline earths, e.g. sodium or magnesium, ammonium or NX 4 + , wherein X is C-_, alkyl. Odier pharmaceutically acceptable salts include organic carboxylic acids such as acetic, lactic, tartaric, malic, isetiiionic, lactobionic, and succinic acids; organic sulfonic acids such as metiianesulfonic, ethanesulfonic, and benzenesulfonic; and inorganic acids such as hydrochloric, sulfuric, phosphoric, and sulfamic acids. Pharmaceutically acceptable salts of a compound having a hydroxyl group include die anion of such compound in combination with a suitable cation such as Na + , NH 4 + , or the like. Sustained release systems suitable for use widi die pharmaceutical compositons of the invention include semi-permeable polymer matrices in the form of films, microcapsules, or the like, comprising polylactides, copolymers of L-glutamic acid and gamma-ethyl-L-glutamate, poly(2-hydroxyetiιyl methacrylate), and like materials. Sustained release systems also include Iiposomally entrapped oligonucleotide clamps, e.g. as described in Liposome Technology, Vol. II, Incorporation of Drugs, Proteins, an Genetic Material (CRC Press). Example 1 Synthesis of Oligonucleotide Clamp having 3' and 5' Cholesterol Binding Moieties for pol and nef genes of HIV A series of oligonucleotide clamps were syndiesized d at have cholesterol moieties attached to eidier a 5' end, a 3' end, or to botii a 3' end and a 5' end. The 3' cholesterol was attached by first constructing a cholesterol-derivatized solid phase support followed by routine oligonucleotide chain extension via phosphoramidite monomers on a conventional automated DNA synthesizer (Applied Biosystems model 394). The 5' cholesterol was attached in die final coupling step of die synti esis by reacting cholesterol chloroformate with the terminal nucleotide having a 5' amino group or by coupling a cholesterol phosphoramidite widi a terminal hydroxyl group, die former method usually giving higher yields. (1) A polymer supported oligonucleotide, lmole scale, with terminal 5'-amino group was treated with 2 ml of a 10% solution of cholesteryl formate in chloroform/diisopropylethylamine (9:1, v:v) for 20 minutes at room temperature. The polymer support was then washed with chloroform and acetonitrile, cleaved and deprotected widi concentrated ammonium (5 hours at 55°C), and purified by reverse phase HPLC. (2) A polymer supported oligonucleotide, 1 μmole scale, with terminal hydroxyl group was treated widi 250 μl of 0.1 M solution of cholesterol phosphoramidite in chloroform and 250 μl of 0.45 M solution of tetrazole in acetonitrile for 10-15 minutes at room temperature. The polymer support was then washed widi acetonitrile, cleaved and deprotected widi concentrated ammonium (5 hours at 55°C), and purified by reverse phase HPLC. Example 2 ATH8 Assay of Oligonucleotide Clamps Specific for pol and nef genes of HIV The oligonucleotides and oligonucleotide clamps listed in die Table I below were syndiesized in accordance widi Example 1 for specific binding to d e following single stranded or double stranded target polynucleotides: 5' AAAAGAAAAGGGGGGA— -3' 3 / TTTTCTTTTCCCCCCT— -5' Double stranded DNA 5' AAAAGAAAAGGGGGGA 3' Single stranded RNA or DNA TABLE I Designation Sequence of Oligonucleotide Clamp" 050A 5 , -CACTTTTC Mc TTTTCCCCCCTCACACTCCCCCC _TTTCTTTTAC-Chol 050B 5 , -Chol-CACTTTTC Me TTTTCCCCCCTCACACTCCCCCC - - -TCTTTTAC-Chol 051 5'-Chol-CACTTTTC Me TTTTC Me C e C Mc C Me C Me C M ^CACACTCCCCCCTTTTCTTTTAC-Chol 052A 5'-CAC _TTTC Me TTTTCCCCCCTCACACT- c Me c e c e c Me c Me c Me τlττC τπAC . cho] 052B 5 , -Chol-CACTTTTC Mc TTTTCCCCCCTCA- CACTC Me C Me C e C Me C Me C Mc TT_TCTTTTAC-Chol 053 5'-Chol-CAC -T-TC M( _TTTGGGGGGTCACACTCCCCCC_ _ _TCTTTTAC-Chol DL015 5'-CAC_ _TTC_TTTGGGGGGTCACACTCCCCCC_ -TTC_TTTAC DL014 5 '-CAC TTTTCTTTTGGGGGGTC AC ACTCCCCCC TTTTCTTTTAC-Chol DL0I3 5'-Chol-CAC -TTTC _TTTGGGGGGTCACACTCCCCCC_ _ _TCTTTTAC-Chol DL021 5 ' -Choi- TTTTCTTTTC AC ACTTTTCTTTTGGGGGGTC AC ACTCCCCCC-Chol DL022 5'-Chol-CACTTTTC -TTTCCCCCCTCACACACTCCCCCC- _TC-TTTAC-Chol DL023 S'-Chol-TTTTCTTTTCACACTTTTCTTTTCCCCCCTCACTCCCCCC-Chol * "Choi " represents cholesterol and C Mc represents 5-methylated cytidine. The melting temperature of the following compounds were determined by computing the half maximum of the 260 nm absorption v. temperature curve, as discribed above: DL015: 39.0°C; DL014: 58°C; DL013: 68.0°C; DL021 : 67.5°C; DL022: 67.5°C; and control (two unconnected oligonucleotides without binding moities having sequences: . e 3'-TTTTCTTTTCCCCCCT-5' and 5'-TTTTC Me TTTT(C 1Vle ) <i - , ): 32.0°C. Each of the compounds of Table I were tested in an ATH8 cell assay to assess the inhibition of the cytopathic effects of HIV infection on the A TH8 cells. The ATH8 assay is described in the following reference: Matsukura, pages 285-304 in Antisense RNA and DNA (Wiley -Liss, New York, 1992). The cytopathic effect examined in the assay was the lytic effect of HIV and/or drug on the ATH8 cells. A measure of the degree of viral activity is the degree to which cells are lysed at the end of a given time period in culture. Protection against lysis is measured by the size of pellet formed after cells are harvested from culture and centrifuged, as described in the references cited above. The results of the assays are summarized in Table II and Figures 3a and 3b. TABLE II Inhibition of Cytotoxic* Inhibition of Cytotoxic* Oligonucleotide Effects at Effects at Clamp 2 mM Concentration 8 mM Concentration Toxicitv** 015 0 0 0 014 + + + + + + 0 013 + + + + 0 021 + + + + 0 022 + + - + 025 + + - + 050A + + + + 0 050B + + - + 051 + + + - + 052A + + + + + 0 052B not tested - + 053 + + 0 * "0" indicates no inhibitory effect observed; number of + 's indicates relative inhibitory effect; "-*-" indicates toxicity at that dose. ** "0" indicates no toxicity observed; " + " indicates some toxicity observed. Figures 3 shows the change in pellet size versus time under different experimental conditions for oligonucleotide clamp 014. Curves 1 and 2 illustrate results from positive and negative controls, i.e. curve 1 illustrates die results for uninfected ATH8 cells without drug (negative control) and curve 2 illustrates d e results for infected ATH8 cells without drug (positive control). Curves 3 and 4 illustrate results from cultures containing 8 μM of oligonucleotide clamp in infected and uninfected ATH8 cells, respectively. Curves 5 and 6 illustrate results from cultures containing 2 μM of oligonucleotide clamp in infected and uninfected ATH8 cells, respectively. Figure 3 shows that clamp 014 has a clear and positive effect at both concentrations without toxicity. Example 3 Synthesis of Two-component Oligonucleotide Clamps widi Cholesterol Binding Moieties The following two-component oligonucleotide clamps were syndiesized by the procedures described above and tested in d e ATH8 assay described above: 5'-Chol-CACTTTTCTTTTGGGGGGTCA-3' 3'-Chol-ACTTTTCTTTTCCCCCCTCAC-5' (DL033/DL034) 5'-Chol-CACTTTTCTTTTGGGGGGTCA-Chol-3' 3'-Chol-ACTTTTCTTTTCCCCCCTCAC-Chol-5' Example 4 Syntiiesis of Oligonucleotide Clamps widi Phosphorodiioate Linkages The following oligonucleotide clamps containing phosphorodiioate linkages were syntiiesized by the procedures described above and tested in die ATH8 assay described above: 5'-Chol-CpsApsCps(Tps) 4 Cps(Tps) 4 (Gps) 6 CpsApsCpsAps- CpsTps(Cps) 6 (Tps) 4 Cps(Tps) 4 CpsAps-Chol-3' 066 5'-Chol-CACTTTTCTTTTGGGGGGT-3' 3'-Chol-ApsCps(Tps) 4 Cps(Tps) 4 (Gps) 6 T-5' 067/DL033 wherein "ps" indicates the presence of a phosphorodiioate linkage. Example 5 Syntiiesis of Oligonucleotide Clamp widi Cholesterol Binding Moieties and a Polyetiiylene Glycol Hinge Region The following oligonucleotide clamp having a non-nucleosidic hinge region is syntiiesized as described above and employing the protected ethyl glycol phosphoramidites disclosed by Durand et al, Nucleic Acids Research, 18: 6353-6359 (1990); and Rumney et al (cited above): 5'-Chol-CAC-π _ CTTTTGGGGGGp(OCH 2 CHJ 5 pTCCCCCCTTTTCTTTTCA-Chol-3 , wherein "p" indicates die presence of a phosphodiester linkage. Example 6 Synthesis of Oligonucleotide Clamp having Bromoacetyl/ phosphorotiiioate Binding Moieties The following oligonucleotide clamp is synthesized by die procedures described above and tested in the ATH8 assay: 5'-BrCH 2 C(= O)NH-CACTTTTCTTTTGGGGGGTCA-Chol-3 ' 3'-SP(=O)(O)AC_ _TTCTTTTCCCCCCTCAC-ChoI-5' BrAc033/PS034 5'-bromoacetylamino oligonucleotides are prepared as follows: 15 μL of 0.4 M N-succinimidyl bromoacetate (e.g. Calbiochem) in N,N-dimethylformamide (DMF) is added to 4.9 A^ units of a 5'-amino-oligonucleotide precursor in 10 mL of 0.2 M sodium borate buffer at room temperature. After about 35 minutes the mixture is diluted (0.5 mL H 2 O), desalted by gel filtration on a NAP-5 column (Pharmacia), purified by reverse phase HPLC (as described below), and again desalted to yield 4 A-^ units of 5'-bromoacetylamino oligonucleotide (elution time for reverse phase HPLC, 17.4 minutes; ion exchange HPLC, 17.4 minutes). Ion exchange HPLC is carried out on a Dionex Omni Pak NA1004x250 mm column at pH 12 (0.001 M NaOH) with a 2%/minute gradient of 1.0 M NaCl in 0.01 M NaOH. Reverse phase HPLC is carried out on a Hypersil ODS 4.6x200 mm column with a 1 %/minute gradient of acetonitrile in 0.03 M triethylammonium acetate buffer, pH 7.0. A 5'-amino-oligonucleotide is prepared as described above. The 3' phosphorodiioate oligonucleotide is formed as described above. Example 7 Synthesis of Oligonucleotide Clamps Consisting of Two Oligonucleotide Moieties in Opposite Orientations widi respect to Hinge Region and/or Binding Moieties The following oligonucleotide clamps were prepared by the procedures described above and as noted below. The 3'-bromoacetyIamino oligonucleotides were prepared similarly to their 5' counterparts, except that a 3'-amino-oIigonucleotide precursor was employed. The 3' phosphorodiioate oligonucleotide is formed as described above. 5'-CACTGGGGGGTTTTGTTTTCA(CH 2 ) 6 NHC(=O)CH 2 Br 5'-ACTCCCCCCTTTTCTTTTCAP(= O)(O)S 064/063 5'-CACTGGGGGGTTTTGTTI CA(3 -X-(3 ACTTTTCTTTTCCCCCCTCA-5' 064/063* wherein X is -(CH 2 ) 6 NHC(=O)CH 2 P(=O)(O " )S-. 5'-Chol-CACTGGGGGGTTTTGTTTTCA(3')-X- (3 ACTTTTCTTTTCCCCCCTCA-Chol-5' 064/063** wherein X is phosphodiester. All three clamps were tested in the ATH8 assays described above. Example 8 Synthesis of Oligonucleotide Clamp Carrying a Fluorescent Label Attached to Hinge Region The following oligonucleotide clamp is synthesized as described above: 5'-Chol-CACTTTTCTTTTGGGGGGCApXpACTCCCCCCTTTTCTTTTCA-Chol-3' wherein "pXp" indicates the presence of a branching linkage or base, e.g. a phosphoramidate linkage, and amino derivatized cytidine, or die like. A phosphoramidate linkage is introduced by carrying out the coupling of one nucleoside as a hydrogen phosphonate monomer then oxidizing d e resulting phosphite linkage widi I 2 and an alkyldiamine, e.g. a hexyldiamine as taught by Agrawal et al, Nucleic Acids Research, 18: 5419-5423 (1990); and Jager et al, Biochemistry, 27: 7237-7246 (1988). This results in a free amine that can be reacted with an activated dye. Preferably, an AminoModifier II branching linkage (DMT-O-CH 2 CH(CH 2 NH-Fmoc)-O-phosphoramidite) (commercially available from Clontech Laboratories, Palo Alto) is introduced into the hinge region. After deprotection and cleavage a free primary amino group is available as an attachment site for a variety of activated fluorescent dyes, e.g. the NHS esters of fluorescein or rhodamine, commercially available from Applied Biosystems (Foster City, CA) as TAMRA-NHS, FAM-NHS, ROX-NHS, and JOE-NHS. Example 9 Synthesis of Oligonucleotide Clamn Carrying a Oligonucleotide Attached to Hinge Region The oligonucleotide clamp of Example 8 is synthesized with die free primary amine in its hinge region. The amine is derivatized with bromoacetyl as described above. Separately, an oligonucleotide is prepared having either a 3' or 5' monophosphorodiioate group, as desired. The bromoacetyl ated clamp and the oligonucleotide are combined in an aqueous solution and frozen as described above. Example 10 Synthesis of Oligonucleotide Clamp Dimer The two oligonucleotide clamps shown below having free amines in tiieir hinge regions are separately synthesized and bromoacetylated as described in Example 2. In a third synthesis, an oligonucleotide, or other linear polymeric unit, is prepared which has a monophosphorothioate group at both its 5' and 3' ends. 5'-Chol-CACTTTTC e TTTTCCCCCCTCAC(pnp)ACTCC- CCCCTTTTCTTTTAC(3 ')-Chol d 5'-choi-'rrrri'i'i rrrri'CAC(pnp)Acrπm ι , ι i"i ι i (3')-choi e SP(0")( = 0)(CH 2 ) 2 p(OCH 2 CH 2 p) l8 (CH 2 ) 2 OP(0 )( = 0)S f wherein "pnp" represents a linkage or monomer containing an bromoacetylamino functionality and "p" is a phosphodiester linkage. After purification d, e, and f are combined in solution and frozen as described above. The oligonucleotide clamp dimer is then purified by gel electrophoresis. SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: Sergei M. Gryaznov (ii) TITLE OF INVENTION: Oligonucleotide clamps having diagnostic and therapeutic applications (iii) NUMBER OF SEQUENCES: 6 (iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: Richard L. Neeley, Ph.D. (B) STREET: Five Palo Alto Square, 4th Floor (C) CITY: Palo Alto (D) STATE: California (E) COUNTRY: USA (F) ZIP: 94306 (v) COMPUTER READABLE FORM: (A) MEDIUM TYPE: 5.25 inch diskette (B) COMPUTER: IBM compatible (C) OPERATING SYSTEM: Windows 3.1/DOS 5.0 (D) SOFTWARE: Microsoft Word for Windows, vers. 2.0 (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: (B) FILING DATE: (C) CLASSIFICATION: (vii) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: 08/087,387 (B) FILING DATE: July 2, 1993 (viii) ATTORNEY/AGENT INFORMATION: (A) NAME: Richard L. Neeley, Ph.D. (B) REGISTRATION NUMBER: 30,097 (C) REFERENCE/DOCKET NUMBER: LYNX-004WO (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: (415) 843-5070 (B) TELEFAX: (415) 857-0663 (2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 16 nucleotides (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: AAAAGAAAAG GGGGGA 16 (2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 42 nucleotides (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: CACTTTTCTT TTCCCCCCTC ACACTCCCCC CTTTTCTTTT AC 42 (2) INFORMATION FOR SEQ ID NO: 3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 42 nucleotides (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: CACTTTTCTT TTGGGGGGTC ACACTCCCCC CTTTTCTTTT AC 42 (2) INFORMATION FOR SEQ ID NO: 4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 42 nucleotides (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: TTTTCTTTTC ACACTTTTCT TTTGGGGGGT CACACTCCCC CC 42 (2) INFORMATION FOR SEQ ID NO: 5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 14 nucleotides (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: TTTTTTTTTT TTCAC 14 (2) INFORMATION FOR SEQ ID NO: 6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 14 nucleotides (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: ACTTTTTTTTTT TTTT 14

Claims: I claim: 1. An oligonucleotide clamp for specifically binding to a target polynucleotide, the oligonucleotide clamp comprising: (a) at least one oligonucleotide moiety capable of specifically binding to the target polynucleotide; and (b) at least one pair of binding moieties, each pair of binding moieties comprising a first binding moiety and a second binding moiety, the first binding moiety being covalently linked to an oligonucleotide moiety and die second binding moiety being covalently linked to an oligonucleotide moiety, wherein a stable covalent or non-covalent linkage is formed between the first binding moiety and the second binding moiety of a pair whenever the first and second binding moieties of the pair are brought into juxtapositon by the specific binding to the target polynucletide of the at least one oligonucleotide moiety to which the first and second binding moieties are attached. 2. The oligonucleotide clamp of claim 1 comprising two components defined by the formulas: X-O r W and Y-O--Z wherein: O. and O 2 are oligonucleotides specific for said target polynucleotide; X and Y are a first pair of binding moieties such that X and Y form a stable covalent or non-covalent linkage or complex whenever they are brought into juxtapositon by the annealing of O* and O 2 to said target polynucleotide; and W and Z are a second pair of binding moieties such that W and Z form a stable covalent or non-covalent linkage or complex whenever they are brought into juxtapositon by the annealing of O* and O 2 to said target polynucleotide. 3. The oligonucleotide clamp of claim 2 wherein O. and O 2 have the same length and are from about 6 to about 40 nucleotides in length. 4. The oligonucleotide clamp of claim 3 wherein one or both of either X and Y or W and Z form a hydrophobic complex. 5. The oligonucletide clamp of claim 4 wherein X and Y are selected from the group consisting of alkanes, fatty acids, fatty alcohols, steroids, waxes, and fat-soluble vitamins. 6. The oligonucleotide clamp of claim 5 wherein X and Y are each a perhydrocyclopentanophenanthrene having from 19 to 30 carbon atoms and from 0 to 6 oxygen atoms. The oligonucleotide clamp of claim 6 wherein X and Y are each cholesterol. 8. The oligonucleotide clamp of claim 5 wherein X, Y, W, and Z are selected from the group consisting of alkanes, fatty acids, fatty alcohols, steroids, waxes, and fat-soluble vitamins. 9. The oligonucleotide clamp of claim 8 wherein X, Y, W, and Z are cholesterol. 10. The oligonucleotide clamp of claim 3 wherein either one or both of X and Y or W and Z form a covalent linkage. 11. The oligonucleotide clamp of claim 10 wherein X is selected from the group consisting of phosphorothioate and phosphorodithioate and wherein Y is selected from the group consisting of haloacyl- or haloalkylamino. 12. The oligonucleotide clamp of claim 11 wherein X and Y are thiophosphoryl. 13. The oligonucleotide clamp of claim 11 wherein W and Z are cholesterol. 14. The oligonucleotide clamp of claim 1 further comprising one or more hinge regions for covalently linking two or more of said oligonucleotide moieties. 15. The oligonucleotide clamp of claim 14 defined by the formula: X-O,-G-O 2 -Y wherein: Oj and O 2 are oligonucleotides specific for said target polynucleotide; G is a hinge region; and X and Y are binding moieties such that X and Y form a stable covalent or non-covalent linkage or complex whenever they are brought into juxtapositon by the annealing of O. and O 2 to said target polynucleotide. 16. The oligonucleotide clamp of claim 15 wherein: O. and O 2 have die same length and are from about 6 to about 40 nucleotides in length: and G is defined by the formula: (M-L) n wherein: n is between 1 and 10; L is a linking group; and M is a straight chain, cyclic, or branched organic molecular structure containing from 1 to 20 carbon atoms and from 0 to 10 heteroatoms selected from the group consisting of oxygen, nitrogen, and sulfur. 17. The oligonucleotide clamp of claim 16 wherein: M is alkyl, alkoxy, alkenyl, or aryl containing from 1 to 16 carbon atoms; heterocyclic having from 3 to 8 carbon atoms and from 1 to 3 heteroatoms selected from the group consisting of oxygen, nitrogen, and sulfur; glycosyl; or nucleosidyl; and L is a phosphorus(V) linking group. 18. The oligonucleotide clamp of claim 17 wherein: M is alkyl, alkoxy, alkenyl, or aryl containing from 1 to 8 carbon atoms; glycosyl; or nucleosidyl; L is selected from the group consisting of phosphodiester, phosphotriester, methyl phosphonate, ethyl phosphonate, phosphorothioate, phosphorodithioate, and phosphoramidate; and n is from 4 to 8. 19. The oligonucleotide clamp of claim 18 wherein X and Y form a hydrophobic complex. 20. The oligonucletide clamp of claim 19 wherein X and Y are selected from the group consisting of alkanes, fatty acids, fatty alcohols, steroids, waxes, and fat-soluble vitamins. 21. The oligonucleotide clamp of claim 20 wherein X and Y are each a perhydrocyclopentanophenanthrene having from 19 to 30 carbon atoms and from 0 to 6 oxygen atoms. 22. The oligonucleotide clamp of claim 21 wherein X and Y are each cholesterol. 23. The oligonucleotide clamp of claim 18 wherein X and Y form a covalent linkage. 24. The oligonucleotide clamp of claim 23 wherein X is selected from the group consisting of phosphorothioate and phosphorodithioate and wherein Y is selected from the group consisting of haloacyl- or haloalkylamino. 25. The oligonucleotide clamp of claim 23 wherein X and Y are thiophosphoryl. 26. The oligonucleotide clamp of claim 23 wherein said oligonucleotide clamp is topolgically linked to said target polynucleotide whenever X and Y form a covalent linkage 27. The oligonucleotide clamp of claim 14 defined by the formula: X-O r G.-O 2 -G 2 -O 3 -Y wherein: O-, O 2 , and O 3 are oligonucleotides specific for said target polynucleotide; Gj and G 2 are hinge regions; and X and Y are binding moieties such that X and Y form a stable covalent or non-covalent linkage or complex whenever they are brought into juxtapositon by the annealing of O-, O 2 , and O 3 to said target polynucleotide. 28. The oligonucleotide clamp of claim 27 wherein: O-, O 2 , and 03 each have a length in the range of about 6 to about 40 nucleotides; and G* and G 2 are defined by the formula: (M-L) n wherein: n is between 1 and 10; L is a linking group; and M is a straight chain, cyclic, or branched organic molecular structure containing from 1 to 20 carbon atoms and from 0 to 10 heteroatoms selected from the group consisting of oxygen, nitrogen, and sulfur. 29. The oligonucleotide clamp of claim 28 wherein: M is alkyl, alkoxy, alkenyl, or aryl containing from 1 to 16 carbon atoms; heterocyclic having from 3 to 8 carbon atoms and from 1 to 3 heteroatoms selected from the group consisting of oxygen, nitrogen, and sulfur; glycosyl; or nucleosidyl; and L is a phosphorus(V) linking group. 30. The oligonucleotide clamp of claim 29 wherein: M is alkyl, alkoxy, alkenyl, or aryl containing from 1 to 8 carbon atoms; glycosyl; or nucleosidyl; L is selected from the group consisting of phosphodiester, phosphotriester, methyl phosphonate, ethyl phosphonate, phosphorothioate, phosphorodidiioate, and phosphoramidate; and n is from 4 to 8. 31. The oligonucleotide clamp of claim 30 wherein X and Y form a hydrophobic complex. 32. The oligonucletide clamp of claim 31 wherein X and Y are selected from the group consisting of alkanes, fatty acids, fatty alcohols, steroids, waxes, and fat-soluble vitamins. 33. The oligonucleotide clamp of claim 32 wherein X and Y are each a perhydrocyclopentanophenanthrene having from 19 to 30 carbon atoms and from 0 to 6 oxygen atoms. 34. The oligonucleotide clamp of claim 33 wherein X and Y are each cholesterol. 35. The oligonucleotide clamp of claim 30 wherein X and Y form a covalent linkage. 36. The oligonucleotide clamp of claim 35 wherein X is selected from the group consisting of phosphorothioate and phosphorodithioate and wherein Y is selected from the group consisting of haloacyl- or haloalkylamino. 37. The oligonucleotide clamp of claim 35 wherein X and Y are thiophosphoryl. 38. The oligonucleotide clamp of claim 35 wherein said oligonucleotide clamp is topolgically linked to said target polynucleotide whenever X and Y form a covalent linkage. 39. A method of inhibiting the expression or function of an endogenous or exogenous gene in a host, the method comprising the step of administering an effective amount of an oligonucleotide clamp specific for the endogenous or exogenous gene or messenger RNA transcribed therefrom. 40. The method of claim 39 wherein said host is a human patient and said endogenous or exogenous gene is a viral gene. 41. The method of claim 40 wherein said viral gene is selected from a genome of a virus selected from the group consisting of herpes virus, cytomegalovirus, human papilloma virus, and human immunodeficiency virus. 42. The method of claim 39 wherein said host is a human patient and said endogenous or exogenous gene is an oncogene. 43. The method of claim 42 wherein said oncogene is selected from the group consisting of myb, myc, bcr-abl, ras, kit, and raf. 44. A pharmaceutical composition comprising a phamaceutical carrier and an oligonucleotide clamp specific for a target polynucleotide whose function or expression directly or indirectly causes a diease. 45. The pharmaceutical composition of claim 44 wherein said oligonucleotide clamp is defined by the formula: X-O--G-O 2 -Y wherein: O. and O 2 are oligonucleotides specific for said target polynucleotide; G is a hinge region; and X and Y are binding moieties such that X and Y form a stable covalent or non-covalent linkage or complex whenever they are brought into juxtapositon by the annealing of O. and O 2 to said target polynucleotide. 46. The pharmaceutical composition of claim 45 wherein said target polynucleotide is a gene selected from a genome of a virus selected from the group consisting of herpes virus, cytomegalovirus, human papilloma virus, and human immunodeficiency virus. 47. A kit for detecting a target polynucleotide, die kit comprising a hybridization buffer and an oligonucleotide clamp specific for the target polynucleotide, the oligonucleotide clamp having a signal generation moiety for generating a detectable signal in the presence of the target polynucleotide.

Inventors: GRYAZNOV SERGEI M, LLOYD DAVID H, GRYAZNOV, SERGEI, M., LLOYD, DAVID, H.
Applicants: GRYAZNOV SERGEI M, LLOYD DAVID H, LYNX THERAPEUTICS INC, GRYAZNOV, SERGEI, M., LLOYD, DAVID, H., LYNX THERAPEUTICS, INC.
Application year: 1994
Main classification: C12Q1
IPC codes: C07H21, C12N15, C12Q1